503 Tumor infiltrating lymphocytes in soft tissue sarcomas upregulate the exhaustion marker TIGIT and are reinvigorated by IL-15 stimulation and TIGIT blockade

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A538-A538
Author(s):  
Sean Judge ◽  
Morgan Darrow ◽  
Steven Thorpe ◽  
Alicia Gingrich ◽  
Edmond O’Donnell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Soft Tissue ◽  
Ex Vivo ◽  
Soft Tissue Sarcomas ◽  
Tumor Infiltrating Lymphocytes ◽  
Cd8 T Cell ◽  
Cell Phenotype ◽  
And Function ◽  
Infiltrating Lymphocytes

BackgroundAlthough the presence and activity of tumor infiltrating lymphocytes (TILs) have been shown to be important factors for survival and response to immunotherapy for multiple cancer types, the benefits of immunotherapy in soft tissue sarcomas (STS) have been limited, and novel approaches are needed. In this study, we sought to characterize the phenotype and function of tumor infiltrating natural killer (NK) and T cells in STS patients and to evaluate clinically relevant strategies to augment TIL function.MethodsUsing both prospectively collected blood and tumor tissue from STS patients undergoing surgical resection (n = 21) and archived specimens (n = 45), we performed flow cytometry and immunohistochemistry to evaluate the extent of peripheral and intratumoral CD3-CD56+ NK and CD8+ T cell phenotype and function as predictors of outcome. We also analyzed TCGA data and the peripheral blood of dogs with spontaneous osteosarcoma receiving inhaled IL-15 on a clinical trial to evaluate the association of CD3-NKp46+ NK and CD8+ T cell activation as well as TIGIT upregulation with outcome. Finally, we stimulated patient PBMCs and TILs ex vivo with IL-15 and a novel human anti-TIGIT antibody to assess the impact of combination therapy on NK and T cell phenotype and function. Parametric and non-parametric statistical tests were used where appropriate. Univariate and multivariate survival analyses were performed by Cox proportional hazards models.ResultsCompared to peripheral expression, intratumoral NK and T cells showed an activated and exhausted phenotype by CD69 and TIGIT, respectively. Ex vivo TIL stimulation with IL-15 further increased markers of activation and function including CD69, Ki67, IFNg, and granzyme B, while increasing expression of exhaustion marker TIGIT. Analysis of a retrospective STS cohort and TCGA STS gene expression confirmed the association of TILs with improved prognosis. Dogs with metastatic osteosarcoma receiving inhaled IL-15 exhibited upregulation of activation markers and TIGIT. In vitro, IL-15 and TIGIT blockade of both peripheral and intratumoral NK cells increased cytotoxicity against sarcoma cell lines and increased expression of degranulation marker CD107a compared to IL-15 alone.ConclusionsTILs are associated with improved survival in STS, and tumor infiltrating NK and T cells show features of both increased activation and increased exhaustion. Tumor-infiltrating NK and T cells respond to IL-15 stimulation, but simultaneously further upregulate TIGIT with the combination of IL-15 and TIGIT blockade showing greatest cytotoxic effects. Overall, our data suggest that the combination of IL-15 and TIGIT blockade is a promising clinical strategy in STS.Ethics ApprovalAll experiments involving human and canine patients were approved by the respective Institutional Review Boards at the University of California, Davis, Schools of Medicine (Protocol #218204-9) and Veterinary Medicine (IACUC #20179).

scholarly journals Analysis of tumor-infiltrating NK and T cells highlights IL-15 stimulation and TIGIT blockade as a combination immunotherapy strategy for soft tissue sarcomas

2020 ◽  
Vol 8 (2) ◽  
pp. e001355
Author(s):  
Sean J Judge ◽  
Morgan A Darrow ◽  
Steve W Thorpe ◽  
Alicia A Gingrich ◽  
Edmond F O'Donnell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Soft Tissue ◽  
T Cell Activation ◽  
Ex Vivo ◽  
Unmet Need ◽  
Soft Tissue Sarcomas ◽  
Cell Phenotype ◽  
Activation Markers ◽  
And Function

PurposeGiven the unmet need for novel immunotherapy in soft tissue sarcoma (STS), we sought to characterize the phenotype and function of intratumoral natural killer (NK) and T cells to identify novel strategies to augment tumor-infiltrating lymphocyte (TIL) function.Experimental designUsing prospectively collected specimens from dogs and humans with sarcomas, archived specimens, and The Cancer Genome Atlas (TCGA) data, we evaluated blood and tumor NK and T cell phenotype and function and correlated those with outcome. We then assessed the effects of interleukin 15 (IL-15) stimulation on both NK and T cell activation and TIGIT upregulation. Finally, we evaluated cytotoxic effects of IL-15 combined with TIGIT blockade using a novel anti-TIGIT antibody.ResultsTILs were strongly associated with survival outcome in both archived tissue and TCGA, but higher TIL content was also associated with higher TIGIT expression. Compared with blood, intratumoral NK and T cells showed significantly higher expression of both activation and exhaustion markers, in particular TIGIT. Ex vivo stimulation of blood and tumor NK and T cells from patients with STS with IL-15 further increased both activation and exhaustion markers, including TIGIT. Dogs with metastatic osteosarcoma receiving inhaled IL-15 also exhibited upregulation of activation markers and TIGIT. Ex vivo, combined IL-15 and TIGIT blockade using STS blood and tumor specimens significantly increased cytotoxicity against STS targets.ConclusionIntratumoral NK and T cells are prognostic in STS, but their activation is marked by significant upregulation of TIGIT. Our data suggest that combined IL-15 and TIGIT blockade may be a promising clinical strategy in STS.

scholarly journals CD8+ T Cells in GCA and GPA: Bystanders or Active Contributors?

2021 ◽  
Vol 12 ◽  
Author(s):  
Rosanne D. Reitsema ◽  
Annemieke M. H. Boots ◽  
Kornelis S. M. van der Geest ◽  
Maria Sandovici ◽  
Peter Heeringa ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Blood Vessels ◽  
Cd8 T Cells ◽  
Current Knowledge ◽  
Adaptive Immune Response ◽  
Cd8 T Cell ◽  
Cell Phenotype ◽  
Functional Features ◽  
And Function

Vasculitis refers to inflammation of blood vessels and can cause a variety of serious complications depending on which vessels are affected. Two different forms of vasculitis are Giant Cell Arteritis (GCA) and Granulomatosis with Polyangiitis (GPA). GCA is the most common form of vasculitis in adults affecting the large arteries and can lead to visual impairment and development of aneurysms. GPA affects small- and medium-sized blood vessels predominantly in the lungs and kidneys resulting in organ failure. Both diseases can potentially be fatal. Although the pathogenesis of GCA and GPA are incompletely understood, a prominent role for CD4+ T cells has been implicated in both diseases. More recently, the role of CD8+ T cells has gained renewed interest. CD8+ T cells are important players in the adaptive immune response against intracellular microorganisms. After a general introduction on the different forms of vasculitis and their association with infections and CD8+ T cells, we review the current knowledge on CD8+ T-cell involvement in the immunopathogenesis of GCA and GPA focusing on phenotypic and functional features of circulating and lesional CD8+ T cells. Furthermore, we discuss to which extent aging is associated with CD8+ T-cell phenotype and function in GCA and GPA.

CD4+CD25+ Marrow Infiltrating Lymphocytes in Myeloma Patients Display an Activated Phenotype and Lack Suppressive Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1741-1741
Author(s):  
Kimberly A. Noonan ◽  
Stephanie Mgebroff ◽  
Paolo Serafini ◽  
Kristen Meckel ◽  
Mark Bonyhadi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Regulatory T Cell ◽  
Cell Phenotype ◽  
Autologous Tumor ◽  
Activation Markers ◽  
Tumor Specificity ◽  
And Function ◽  
Infiltrating Lymphocytes

Abstract We have previously shown that marrow infiltrating lymphocytes (MILs) from myeloma patients proliferate upon stimulation with anti-CD3/CD28 beads more efficiently and exhibit greater specificity to autologous tumor than their peripheral blood lymphocyte (PBL) counterparts. In attempting to dissect the mechanisms responsible for greater tumor specificity of MILs, we examined the frequency and function of the putative CD4+/CD25+ regulatory T cells (Tregs) in both compartments. Phenotypic and functional differences in the CD4+/CD25+ populations were examined in MILs and PBLs from 12 multiple myeloma patients and 3 normal donors. CD4+/CD25+ cells were stained for the suppressive marker: FoxP3; or the activation markers: CD40L, CD71 (transferrin receptor). CD4+/CD25+ MILs of myeloma patients are CD25lo, FoxP3lo and predominantly express activation markers. They expand more readily upon anti-CD3/CD28 stimulation, produce high amounts of the Th1 cytokines: IFNγ and IL-2, and low amounts of IL-10. Importantly, they fail to suppress the tumor specific T cell proliferation. These findings are all consistent with an activated T cell phenotype. In contrast, CD4+/CD25+ cells from PBLs of myeloma patients are CD25hi, FoxP3+, primarily produce IL-10 and IL-4 and markedly suppress T cell expansion in a tumor specific assay, suggestive of a regulatory T cell phenotype. Although MILs possess a lower number of FoxP3+ cells as compared to PBLs, the higher MFI may indicate the presence of a small yet highly regulatory T cell population. Interestingly, MILs from normal donors possess a more regulatory phenotype then their myeloma counterparts as determined by high FoxP3 and low CD40L and CD71 expression as well as reduced expansion upon activation. Taken together, these data underscore major differences in the immuno-inhibitory pathways present in blood as compared to marrow. Notably, MILs of myeloma patients demonstrate a paucity of Tregs. These differences may explain the disparities seen in the tumor-specificity of T cells from these two compartments. The ability to expand a T cell population with fewer endogenous Tregs and heightened tumor-specificity may have significant implications for the implementation of adoptive immunotherapy. %CD25 (MFI) %FoxP3 (MFI) %CD40L %CD71 Fold Expansion Ability to Suppress Myeloma CD4/CD25 MILs 4.7 (29.6) 2.2 (2932.1) 88.8 90.1 12.2 − CD4/CD25 PBLs 19.1 (115.2) 52.3 (163.7) 15.7 19.6 2.5 +++++ Normal CD4/CD25 MILs 14.0 (56.8) 70.1 (82.7) 37.3 6.4 3.3 +++ CD4/CD25 PBLs 24.0 (19.4) 30.9 (261.8) 12.1 4.2 4.0 ++

498 Downregulation of CD5 in CD8+ T tumour-infiltrating lymphocytes associates with increased level of activation and exhaustion

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A533-A533
Author(s):  
Faizah Alotaibi ◽  
Mark Vincent ◽  
Weiping Min ◽  
James Koropatnick
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Ex Vivo ◽  
Cd8 T Cell ◽  
Tcr Signaling ◽  
Peripheral Organs ◽  
Infiltrating Lymphocytes

BackgroundCD5, a member of the scavenger receptor cysteine-rich superfamily, is a marker for T cells and a subset of B cells (B1a). CD5 associates with T-cell and B-cell receptors and impair TCR signaling1 2 and increased CD5 is an indication of B cell activation. Furthermore, CD5 levels on CD8+ T cell splenocytes were significantly increased after TCR/CD3 stimulation using ex vivo treatment with anti-CD3/anti-CD28 MAbs compared to non-stimulated CD8+ T splenocytes.3 Previous studies have shown a correlation between CD5 and anti-tumour immunity where CD5 knockout mice inoculated with B16F10 melanoma cells had delayed tumour growth compared to wild type mice.4 In tumour-infiltrating lymphocytes (TILs) isolated from lung cancer patients, CD5 levels were negatively correlated with anti-tumour activity and tumour-mediated activation-induced T cell death,5 suggesting that CD5 could impair activation of anti-tumour T cells. However, the correlation between CD5 level expression and T cell activation and exhaustion in the tumour microenvironment and in peripheral organs is ill-defined and requires further investigation.MethodsWe determined CD5 levels in T cell subsets in different organs in mice bearing syngeneic 4T1 breast tumour homografts and assessed the relationship between CD5 and increased CD69 and PD-1 (markers of T cell activation and exhaustion) by flow cytometry.ResultsWe report that T cell CD5 levels were higher in CD4+ T cells than in CD8+ T cells in 4T1 tumour-bearing mice, and that high CD5 levels on CD4+ T cells were maintained in peripheral organs (spleen and lymph nodes). However, both CD4+ and CD8+ T cells recruited to tumours had reduced CD5 compared to CD4+ and CD8+ T cells in peripheral organs. In addition, CD5highCD4+ T cells and CD5highCD8+ T cells from peripheral organs exhibited higher levels of activation and associated exhaustion compared to CD5lowCD4+ T cell and CD5lowCD8+ T cell from the same organs. Interestingly, CD8+ T cells among TILs and downregulated CD5 were activated to a higher level, with concomitantly increased exhaustion markers, than CD8+CD5+ TILs.ConclusionsThus, differential CD5 levels among T cells in tumours and lymphoid organs can be associated with different levels of T cell activation and exhaustion, suggesting that CD5 may be a therapeutic target for immunotherapeutic activation in cancer therapy.AcknowledgementsThe author thanks Rene Figueredo and Ronak Zareardalan for their assistance in animal workEthics ApprovalThis study was approved by the Animal Use Subcommittee of the University of Western OntarioReferencesAzzam HS, et al., Fine tuning of TCR signaling by CD5. The Journal of Immunology 2001. 166(9): p. 5464–5472.Voisinne GA, Gonzalez de Peredo and Roncagalli R. CD5, an undercover regulator of TCR signaling. Frontiers in Immunology 2018;9:p. 2900.Alotaibi, F., et al., CD5 blockade enhances ex vivo CD8+ T cell activation and tumour cell cytotoxicity. European journal of immunology 2020;50(5): p. 695–704.Tabbekh, M., et al., Rescue of tumor-infiltrating lymphocytes from activation-induced cell death enhances the antitumor CTL response in CD5-deficient mice. The Journal of Immunology, 2011. 187(1): p. 102–109.Dorothée, G., et al., In situ sensory adaptation of tumor-infiltrating T lymphocytes to peptide-MHC levels elicits strong antitumor reactivity. The Journal of Immunology 2005;174(11): p. 6888–6897.

Detection of virus-specific T cells and CD8+ T-cell epitopes by acquisition of peptide–HLA-GFP complexes: analysis of T-cell phenotype and function in chronic viral infections

10.1038/nm845 ◽  
2003 ◽  
Vol 9 (4) ◽  
pp. 469-475 ◽  
Author(s):  
Utano Tomaru ◽  
Yoshihisa Yamano ◽  
Masahiro Nagai ◽  
Dragan Maric ◽  
Previn T.P. Kaumaya ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Viral Infections ◽  
Cd8 T Cell ◽  
Cell Phenotype ◽  
T Cell Epitopes ◽  
Chronic Viral Infections ◽  
And Function

scholarly journals The future of immunotherapy

2020 ◽  
Author(s):  
Cornelis J M Melief
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
Checkpoint Control ◽  
Checks And Balances ◽  
Drug Conjugates ◽  
Cell Immunity ◽  
Anti Cd20 ◽  
Infiltrating Lymphocytes

Abstract During the last two decades two main schools of modern immunotherapy have come to the forefront. The chimeric anti-CD20 antibody rituximab that was introduced for the treatment of refractory follicular lymphoma in 1998 was one of the first examples of the school of passive immunotherapy. Subsequently major and ever more costly efforts were spent on the development of blockbuster monotherapies including other monoclonal but also bispecific antibodies of highly defined specificity and subclass, antibody-drug-conjugates (ADCs), as well as ex vivo expanded tumor infiltrating lymphocytes, CAR-transduced T cells, and TCR-transduced T cells. On the other hand there is the school that works towards active induction of patient B- or T-cell immunity against antigens of choice, or active tolerance against pathogenic allergens, auto-antigens or allo-antigens. Stradled in between these two approaches is treatment with blockers of T cell checkpoint control, which releases the brakes of T cells that have already responded to antigen. Extensive and detailed insight into the cellular and molecular interactions that regulate specific immune responses is indispensable in order to be able to optimize efficacy and rule out treatment related toxicity. This applies to all types of immunotherapy. Our knowledge of the checks and balances in the immune system is still increasing at an unprecedented pace, fostering ever more effective and specific (combination) immunotherapies and offering a rich harvest of innovative immunotherapies in the years ahead.

Expression of LAG-3 by tumor-infiltrating lymphocytes is coincident with the suppression of latent membrane antigen–specific CD8+ T-cell function in Hodgkin lymphoma patients

Blood ◽  
2006 ◽  
Vol 108 (7) ◽  
pp. 2280-2289 ◽  
Author(s):  
Maher K. Gandhi ◽  
Eleanore Lambley ◽  
Jaikumar Duraiswamy ◽  
Ujjwal Dua ◽  
Corey Smith ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Hodgkin Lymphoma ◽  
Cell Function ◽  
T Cell Responses ◽  
Tumor Infiltrating Lymphocytes ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Infiltrating Lymphocytes

AbstractIn Hodgkin lymphoma (HL), the malignant Hodgkin Reed-Sternberg (HRS) cells constitute only 0.5% of 10% of the diseased tissue. The surrounding cellular infiltrate is enriched with T cells that are hypothesized to modulate antitumor immunity. We show that a marker of regulatory T cells, LAG-3, is strongly expressed on infiltrating lymphocytes present in proximity to HRS cells. Circulating regulatory T cells (CD4+ CD25hi CD45 ROhi, CD4+ CTLA4hi, and CD4+ LAG-3hi) were elevated in HL patients with active disease when compared with remission. Longitudinal profiling of EBV-specific CD8+ T-cell responses in 94 HL patients revealed a selective loss of interferon-γ expression by CD8+ T cells specific for latent membrane proteins 1 and 2 (LMP1/2), irrespective of EBV tissue status. Intratumoral LAG-3 expression was associated with EBV tissue positivity, whereas FOXP3 was linked with neither LAG-3 nor EBV tissue status. The level of LAG-3 and FOXP3 expression on the tumor-infiltrating lymphocytes was coincident with impairment of LMP1/2-specific T-cell function. In vitro pre-exposure of peripheral blood mononuclear cells to HRS cell line supernatant significantly increased the expansion of regulatory T cells and suppressed LMP-specific T-cell responses. Deletion of CD4+ LAG-3+ T cells enhanced LMP-specific reactivity. These findings indicate a pivotal role for regulatory T cells and LAG-3 in the suppression of EBV-specific cell-mediated immunity in HL.

scholarly journals Maximizing cancer therapy via complementary mechanisms of immune activation: PD-1 blockade, neoantigen vaccination, and Tregs depletion

2021 ◽  
Vol 9 (11) ◽  
pp. e003480
Author(s):  
Anna Morena D'Alise ◽  
Guido Leoni ◽  
Maria De Lucia ◽  
Francesca Langone ◽  
Linda Nocchi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Infiltrating Lymphocytes ◽  
Cell Phenotype ◽  
Cancer Immunity ◽  
Cell Immunity ◽  
Combination Approach ◽  
Vectored Vaccine ◽  
Cure Rates ◽  
Infiltrating Lymphocytes

BackgroundA number of different immune pathways are involved in the effective killing of cancer cells, collectively named as the ‘Cancer Immunity Cycle’. Anti-PD-1 checkpoint blockade (CPB) therapy is active on one of these pathways and reinvigorates anticancer T cell immunity, leading to long-term responses in a limited fraction of patients with cancer. We have previously shown that neoantigens-based adenovirus vectored vaccine in combination with anti-PD-1 further expands pre-existing anticancer immunity and elicits novel neoantigen-specific T cells thereby increasing efficacy to 50% of tumor clearance in mice. Here we added a third component to the CPB plus vaccine combination, which is able to modify the suppressive tumor microenvironment by reducing the number of tumor-infiltrating regulatory T cells (Tregs), as strategy for improving the therapeutic efficacy and overcoming resistance.MethodsThe antitumor efficacy of anti-PD-1, neoantigen vaccine and Treg modulating agents, either Bempegaldesleukin (BEMPEG: NKTR-214) or an anti-CTLA-4 mAb with Treg-depleting activity, was investigated in murine tumor models. We evaluated tumor growth in treated animals, neoantigen-specific T cells in tumors, tumor-infiltrating lymphocytes (TILs) and intratumoral Tregs.ResultsThe addition of BEMPEG or anti-CTLA-4 to the combination of vaccine and anti-PD-1 led to complete eradication of large tumors in nearby 100% of treated animals, in association with expansion and activation of cancer neoantigen-specific T cells and reduction of tumor-infiltrating Tregs.ConclusionThese data support the notion that the integrated regulation of three steps of the cancer immunity cycle, including expansion of neoantigen-specific T cells, reversal of the exhausted T cell phenotype together with the reduction of intratumoral Tregs may represent a novel rationally designed drug combination approach to achieve higher cure rates.

scholarly journals The tumor microenvironment as a metabolic barrier to effector T cells and immunotherapy

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Aaron R Lim ◽  
W Kimryn Rathmell ◽  
Jeffrey C Rathmell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Cell Metabolism ◽  
Tumor Infiltrating Lymphocytes ◽  
T Cell Signaling ◽  
Waste Products ◽  
Cancer Cell Metabolism ◽  
And Function ◽  
Infiltrating Lymphocytes

Breakthroughs in anti-tumor immunity have led to unprecedented advances in immunotherapy, yet it is now clear that the tumor microenvironment (TME) restrains immunity. T cells must substantially increase nutrient uptake to mount a proper immune response and failure to obtain sufficient nutrients or engage the appropriate metabolic pathways can alter or prevent effector T cell differentiation and function. The TME, however, can be metabolically hostile due to insufficient vascular exchange and cancer cell metabolism that leads to hypoxia, depletion of nutrients, and accumulation of waste products. Further, inhibitory receptors present in the TME can inhibit T cell metabolism and alter T cell signaling both directly and through release of extracellular vesicles such as exosomes. This review will discuss the metabolic changes that drive T cells into different stages of their development and how the TME imposes barriers to the metabolism and activity of tumor infiltrating lymphocytes.

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